The proper and harmonious expression of a large number of genes is a critical component of normal growth and development and the maintenance of proper health. Disruptions or changes in gene expression are responsible for many diseases. Using traditional methods to assay gene expression, researchers were able to survey a relatively small number of genes at a time. Microarrays allow scientists to analyze expression of many genes in a single experiment quickly and efficiently. A microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated.
DNA arrays are commonly used to study gene expression. In this type of study, mRNA is extracted from a sample (for example, blood cells or tumor tissue), converted to complementary DNA (cDNA) and tagged with a fluorescent label. In a typical microarray experiment, cDNA from one sample (sample A) is labeled with a first dye that fluoresces in the red and cDNA from another sample (sample B) is labeled with a different dye that fluoresces in the green. The fluorescent red and green cDNA samples are then applied to a microarray that contains DNA fragments (oligonucleotides) corresponding to thousands of genes. If a DNA sequence probe is present on the microarray and its complement is present in one or both samples, the sequences bind, and a fluorescent signal can be detected at the specific spot on the array, where the DNA sequence probe is located. The signals are generally picked up using a “scanner” which creates a digital image of the array. The red to green fluorescence ratio in each spot reflects the relative expression of a given gene in the two samples. The result of a gene expression experiment is referred to as a gene expression “profile” or “signature”.
This technology, though widely used, is not without its problems. Almost every procedure in the methodology is a potential source of fluctuation leading to a lot of noise in the system as a whole. The major sources of fluctuations to be expected are in mRNA preparation, reverse transcription leading to cDNA of varying lengths, systemic variation in pin geometry, random fluctuations in spot volume, target fixation, slide non-homogeneities due to unequal distribution of the probe, hybridization parameters and non-specific hybridization. Some of the errors mentioned above can be minimized by performing replicates of experiments or by using a flipped dye design.
Biological replicates are arrays that each use RNA samples from different individual organisms, pools of organisms or flasks of cells, but yet compare the same treatments or control/treatment combinations. Technical replicates are arrays that each use the same RNA samples and also the same treatment. Thus, in this setting, the only differences in measurements are due to technical differences in array processing. The rationale for the flipped dye design is that it allows for the estimation and removal of gene specific dye effects. These dye effects have been shown to be reproducible across independent arrays by the use of Control vs. Control arrays. Any deviation from a ratio of 1 in these arrays is due to either dye effect or residual error. However, none of these methods will account accurately for chip manufacturing error.
Therefore, there remains a need for the development of improved microarray technologies, and particularly technologies that allow researchers to control for errors and/or to normalize signals.